Pharmaceutical formulations and uses thereof in the treatment of female sexual dysfunction

ABSTRACT

The present invention relates to the use of a combination of a PDE5-inhibitor and testosterone for the preparation of a medicament for the treatment of Female Sexual Dysfunction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/620,576filed on 12 Jun. 2017, now allowed, which is a divisional of U.S.application Ser. No. 14/866,547 filed on 25 Sep. 2015, now U.S. Pat. No.9,700,566 issued on 11 Jul. 2017, which is a divisional of U.S.application Ser. No. 13/529,813 filed on 21 Jun. 2012, now U.S. Pat. No.9,192,669 issued 24 Nov. 2015, which is a divisional of U.S. applicationSer. No. 11/595,778 filed 10 Nov. 2006, now U.S. Pat. No. 8,227,453issued 24 Jul. 2012, which is a continuation of PCT applicationPCT/NL2005/000355 having an international filing date of 11 May 2005,which claims benefit of European patent application Nos. 04076402.9filed 11 May 2004, 04078033.0 filed 4 Nov. 2004, 04078380.5 filed 13Dec. 2004, 04078381.3 filed 13 Dec. 2004, and 04078455.5 filed 21 Dec.2004. The contents of the above patent applications are incorporated byreference herein in their entirety.

TECHNICAL FIELD

The invention relates to the field of female sexual dysfunction. Itspecifically relates to the influence of the combination of testosteroneor an analogue thereof and a PDE5 inhibitor (such as sildenafil,vardenafil or tadalafil) on sexual health in female subjects with FemaleSexual Dysfunction (such as Female Sexual Arousal Disorder (FSAD) orFemale Sexual Desire Disorder (FSDD)).

BACKGROUND ART

Female Sexual Dysfunction (FSD) refers to various disturbances orimpairments of sexual function, including a lack of interest in sexualactivity, repeated failure to attain or maintain sexual excitement,inability to attain an orgasm following sufficient arousal. A recentstudy estimated that 43% of women suffer from sexual dysfunction in theUSA¹. Low sexual desire (22% prevalence) and sexual arousal problems(14% prevalence) belong to the most common categories of sexualdysfunction of women. These categories are convenient in providingworking definitions and an accepted lexicon for researchers andtherapists. However, it may be incorrect to assume that these disordersare fully independent of each other. Both case studies andepidemiological studies demonstrate that these disorders can overlap andmay be interdependent. In some cases, it may be possible to identify theprimary disorder that led to the others, but in many cases, this may beimpossible.

For the treatment of female sexual disorder a number of differenttreatments, with greater or lesser degrees of success have beensuggested and applied. These treatments have either not been completelysuccessful or the side effects are hardly acceptable. The presentinvention provides a new combination of therapeutic substances, given ina particular dosage scheme, which combination is effective and does nothave serious side effects.

Thus the invention provides the use of a combination of a PDE5-inhibitorand testosterone or an analogue thereof, in the preparation of amedicament for the treatment of female sexual dysfunction. According tothe invention, although it is not considered bound by theory, an effecton the central nervous system and the peripheral system are required,whereby the signal to the central system is provided by testosterone oran analogue thereof (having the same kind of activity) and theperipheral signal is provided by a PDE5-inhibitor. According to theinvention the level of free testosterone should be a peak plasma levelof free testosterone at least of about 0.010 nmol/L, which willtypically occur about 20 minutes after administration of thetestosterone. According to the invention, the effect of the peak plasmalevel of at least 0.010 nmol/l of free testosterone is to be reached atabout the same time as the effect of the PDE-5 inhibitor. For an optimaleffect it is desired that the peak effect of both compounds coincide.However, even if the peak effects only overlap partly, this stillresults in the desired effect (treatment of FSD). There is a time lagfor the effect of testosterone (or the analogue) of about 3-6 (morespecifically around 3-4.5) hours, in particular around 4 hours.PDE5-inhibitors such as vardenafil and sildenafil typically reach theirpeak plasma concentration (which should be at least 35 ng/ml forsildenafil, 2 μg/L for vardenafil and 40 μg/L for tadalafil) after about1 hour after administration and thus the two pharmaceuticals arepreferably presented as a kit of parts with instructions about theadministration, or are packaged in one capsule or formula withdifferential release properties for the two compounds.

Testosterone in the circulation is typically bound by SHBG (steroidhormone binding globulin) and by albumin. It is important that the peakplasma level of testosterone as defined in the present invention ispresent and calculated as free testosterone, so a fraction not bound byalbumin and SHBG. Thus the dose of testosterone given should be highenough to saturate the albumin and SHBG (i.e the concentration oftestosterone must be high enough to overcome complete binding oftestosterone by SHBG or albumin), or another way of avoiding binding toalbumin or SHBG must be designed, such as the use of a competitor forthe testosterone binding site on SHBG.

Testosterone is preferably given in a formulation wherein there is ashort high peak in the blood circulation of the subject to which it isadministered. The invention therefore provides a use, wherein thetestosterone or an analogue thereof is provided in the form of asublingual formulation, preferably a sublingual formulation comprisingcyclodextrins as carrier. A typical example of such a formulation isgiven in hydroxypropyl-beta cyclodextrin, but other beta cyclodextrinsand other usual excipients, diluents and the like are within the skillof the art for preparing a formulation comprising testosterone or ananalogue thereof, which releases essentially all of the testosteronewithin one short burst. Said burst will typically be within a short timeinterval (for example within 60-120 seconds, more preferably within 60seconds) upon administration, leading to blood peak levels oftestosterone about 15-20 minutes later. In a preferred embodiment, thepharmaceutical is designed for sublingual administration and even morepreferred said composition comprises cyclodextrin such ashydroxypropyl-beta cyclodextrin. A typical example of a preparedtestosterone sample (for 0.5 mg of testosterone) consists of 0.5 mgtestosterone, 5 mg hydroxypropyl-betacyclodextrines (carrier), 5 mgethanol, and 5 ml water, but each of the amounts of these substancesmight be higher or lower.

Of course the pharmaceutical preparation comprising a PDE5-inhibitorshould also be designed to give a peak plasma level at about the timewhen the testosterone effect is maximal. Such compositions are withinthe skill of the art, a typical example for oral administration is givenin vardenafil HCl which is designated chemically as piperazine,1-[[3-(1,4-dihydro-5-methyl-4-oxo-7-propylimidazo[5,1-f][1,2,4]triazin-2-yl)-4-ethoxyphenyl]sulfonyl]-4-ethyl-,monohydrochloride. In addition to the active ingredient, vardenafil HCl,each tablet contains microcrystalline cellulose, crospovidone, colloidalsilicon dioxide, magnesium stearate, hypromellose, polyethylene glycol,titanium dioxide, yellow ferric oxide, and red ferric oxide. An otherexample is given in sildenafil citrate which is chemically designated as1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1Hpyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazinecitrate. In addition to the active ingredient, sildenafil citrate, eachtablet contains the following ingredients: microcrystalline cellulose,anhydrous dibasic calcium phosphate, croscarmellose sodium, magnesiumstearate, hydroxypropyl methylcellulose, titanium dioxide, lactose,triacetin, and FD & C Blue #2 aluminum lake. An other example is givenin tadalafil which is chemically designated aspyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione,6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-, (6R,12aR)-.In addition to the active ingredient, tadalafil, each tablet containsthe following ingredients: croscarmellose sodium, hydroxypropylcellulose, hypromellose, iron oxide, lactose monohydrate, magnesiumstearate, microcrystalline cellulose, sodium lauryl sulfate, talc,titanium dioxide, and triacetin.

It is clear, that preferably the (peak) effect of PDE5-inhibitor as wellas the (peak) effect of testosterone coincide (completely). It ishowever noted that if the peak effect of testosterone and of thePDE5-inhibitor only partly overlap this still results in the desiredeffect. When the testosterone is provided such that it essentiallyreleases all of the testosterone within one short burst to a femalesubject, the PDE5-inhibitor is preferably provided such that it resultsin a peak plasma concentration at least 3 hours after the administrationof testosterone. Even more preferred, the PDE5-i effect is present3.5-5.5 hours after the intake of testosterone. It is clear that theexact time of PDE5-inhibitor administration is dependent on the type offormulation used. If the PDE5-inhibitor formulation is released shortlyafter administration, it is of no use to provide it at the same time asthe testosterone is provided, because there will be hardly any overlapof effect. If it takes some time before the PDE5-inhibitor is availablefrom the used formulation, for example 3 to 4 hours, it can be/isadministrated at the same time the testosterone is administrated.

For the present invention the routes of administration of choice arethose which are the least invasive. Motivation for sexual behaviourshould not be negatively influenced by invasive routes ofadministration. Because there is a time lag in the effect oftestosterone, the two drugs necessary for a central effect and aperipheral effect cannot be administered at the same time (unless theadministration of the PDE5-inhibitor is designed such that the drug isreleased after 3.5-5.5 hours after administration). The inventiontherefore provides a kit of parts comprising at least one pharmaceuticalcomposition comprising testosterone or an analogue thereof and at leastone pharmaceutical composition comprising a PDE5-inhibitor, whereby saidcomposition comprising testosterone is designed to release alltestosterone essentially immediately (for example within 60 seconds) atthe target site. Said kit preferably contains instructions to use apharmaceutical composition comprising testosterone 3.5-5.5 hours priorto sexual activity and a pharmaceutical composition comprising aPDE5-inhibitor 1-2 hours prior to sexual activity. The kit of parts maycomprise a sublingual formulation of testosterone or an analogue thereofand a tablet or another formulation comprising a PDE5-inhibitor. Thepreferred PDE-5 inhibitors are sildenafil, vardenafil or tadalafil. Theamount of testosterone per pharmaceutical composition comprisingtestosterone is at least 0.3 mg testosterone and at most 2.5 mgtestosterone. Higher or lower doses may be necessary depending on thealbumin and SHBG levels and the weight of the subject to be treated. Thepharmaceutical composition comprising a PDE5-inhibitor comprises atleast 25 mg sildenafil (or 5 mg vardenafil, or 5 mg tadalafil) and atmost 100 mg sildenafil (or 20 mg vardenafil, or 20 mg tadalafil), orcomparable dosages of other PDE5-inhibitors. Again these doses may varywith the weight of the patient. For the reasons already outlined above,a kit according to the invention may further comprise a compound capableof competing with testosterone or an analogue thereof for SHBG binding).

In a preferred embodiment, the testosterone analogue is a precursor ormetabolite of testosterone. In case a precursor of testosterone is used,the kit further comprises instructions to (if necessary) increase thetime period of 3.5-5.5 hours by adding the time which is needed toconvert the precursor into testosterone. In case a metabolite oftestosterone is used the time period of 3.5-5.5 hours is shortened.

In order to further enhance the effects of the kit of parts of theinvention said kit may further comprise means for cognitiveinterventions and stimulation. Such information may be present on anydata carrier (paper, CD, DVD), passive or interactive, or it may be alink to a website at least partially designed for the purpose of saidcognitive stimulation. Sometimes it is preferred to present saidcognitive stimulatory information subconsciously e.g. subliminally.

To further enhance the effects of the kit of the present invention asubstance may be added to said kit which stimulates the mesolimbicdopaminergic pathway in the subject. This pathway is concerned with arelatively different kind of reward system which helps providing anincrease in rewardseeking involved in sexual behaviour. Examples of suchcompounds are Apomorphine, a dopamine D2 agonist; Aripiprazol, a partialdopamine D2 agonist; Pergolide, a nonselectieve dopamine (DA) agonist;Pramipexole, a new dopamine receptor agonist with preference for D3compared to D2 and D4 receptors; Bromocriptine, a nonselectieve dopamine(DA) agonist; Ropinirole hydrochloride, a non-ergoline dopamine agonistwith a relatively high in vitro specificity and full intrinsic activityat the D₂ and D₃ dopamine receptor subtypes; it binds with higheraffinity to D₃ than to D₂ or D₄ receptor subtypes; Roxindole, a potent(autoreceptor)-“selective” D3 dopamine agonist; Cabergoline, a dopamineD2 agonist; Lisuride, a nonselectieve dopamine (DA) agonist, and theautoreceptor antagonists; (+)-AJ 76, a D3-preferring, dopamine (DA)autoreceptor antagonist; (+)-UH232, a stimulant of dopaminergictransmission, which may preferentially antagonize autoreceptors ofdopamine nerve terminals, as well as the reuptake blockers; Bupropion,an inhibitor of the neuronal uptake of norepinephrine, serotonin anddopamine; Amineptine, a (relatively) selective dopamine reuptakeinhibitor; GBR 12909 (vanoxerine), a dopamine reuptake inhibitor; andAmantadine; a NMDA receptor antagonist and dopamine reuptake inhibitor.

To further enhance the effects of the kit of the present invention asubstance is (optionally) added which inhibits the central andperipheral adrenergic tone, i.e. inhibits or dampens central andperipheral extracellular norepinephrine concentrations. Activation ofalpha 2-receptors located in the central nervous system results ininhibition of sympathetic tone. Examples of such compounds areclonidine, an alpha 2 agonist; imidazoline, a partial alpha 2 agonist;and dexmedetomidine, an alpha 2 agonist.

The kit of parts is useful for any individual suffering from any form ofFSD, be it through psychological or physiological causes or combinationsthereof. It is thus also useful for subjects having FSD because of othermedicines and/or drugs—such as SSRI's-; subjects suffering fromhypogonadism, etcetera.

Low sexual desire, sexual arousal problems and hampered orgasm arecandidates for psychopharmacological treatment. These categories ofsexual problems are also linked to three (transitional and overlapping)phases of the human sexual response (sexual desire, sexual arousal andorgasm), which are regulated by relatively independent neurotransmitterfunctions. Traditionally, motivated behaviours have been divided intoappetitive and consummatory components. Activities aimed at obtainingreward and satisfaction belong to the appetitive component. Thefundamental appetitive motivational process is an intrinsic brainfunction, and is especially related to the predictive value of stimulifor reward. Processing of motivationally relevant information (i.e.stimuli predicting reward) causes an increase in activity of themeso-accumbens dopaminergic (DA) system (i.e. DA neurons of the ventraltegmental area (VTA) innervating the nucleus accumbens (NAS)), acomponent of the mesolimbic dopamine system. The activity of this systemis increased during flexible approach behaviour when anticipating rewardrelated to copulation². Increasing activity in these dopaminergicpathways facilitates sexual motivation, in particular anticipatorysexual behavior³. Aripiprazole is, among others, an example of a drugwhich influences dopaminergic pathways, and which may be used incombination with testosterone or an analogue thereof and aPDE5-inhibitor to affect sexual motivation and behaviour. Aripiprazol isa high-affinity partial agonist of the dopamine D2 receptor andserotonin 5-HT1a receptor, and antagonist of the 5-HT2a receptor.Aripiprazol is described as a dopamine system stabilizer which is due toits partial agonistic actions at the D2 receptor, especially thepresynaptic D2 receptors, for which it has higher affinity. Stimulationof autoreceptors located on dopamine nerve terminals results in aninhibition of dopamine synthesis and release. Thus, in a lowdopaminergic state of the meso-accumbens DA system, aripiprazol wouldantagonize presynaptic D2 receptors, freeing the NAS-projecting DAnuclei in the VTA from autoinhibition. The medial prefrontal cortex(mPFC) mediates behavioural inhibition. Dopamine in the mPFC plays animportant role in behavioural inhibition. Illustrative of mPFC-DA'sinhibitory role is the inhibition of the meso-accumbens DA system; highextracellular concentrations of mPFC-DA inhibit meso-accumbal DAactivity, and low extracellular concentrations of mPFC-DA activatemeso-accumbal DA activity through disinhibition. It is thereforeconceivable that a dopaminergic role in FSD is not restricted tomeso-accumbal DA, but extendable to mPFC-DA, where symptoms of FSD areenhanced with high activity of mPFC-DA, albeit via inhibition ofaccumbal DA or via inhibition of other cognitive or emotional factorsinvolved in FSAD. The partial agonistic action of aripiprazol will thenhave a positive effect on alleviation of FSD (symptomatology) throughagonism of presynaptic D2 receptor in the mPFC, thereby inhibiting DArelease in this area. Anticipating sexual reward will produce arousal ofthe genitalia, in which at least three key neurotransmitters areinvolved: acetylcholine, norepinephrine and nitric oxide. Acetylcholineand nitric oxide both promote erections in men and lubrication andswelling in women. Norepinephrine inhibits erections in men andlubrication and swelling in women. Orgasm, the consummatory phase ofhuman sexual response is facilitated by descending spinal noradrenergicfibers and innervation of the genitalia, and inhibited by descendingspinal serotonergic fibers.

Testosterone in Women

In many mammalian species, female sex steroids are necessary for theexpression of female sexual behavior. As a result the capability forcopulation in these animals is limited to the period of ovulation^(4,5).Higher primates—like humans—show sexual intercourse outside theperiovulatory period. For these animals it has been suggested thattestosterone is involved in female sexual behavior⁶ . The disappearanceof testosterone following ovariectomy and adrenalectomy is accompaniedby a complete loss of libido^(7,8), while substitution of this steroidmaintains sexual desire and fantasies after surgical menopause⁹.

Testosterone, Exposure to Sexual Cues and Vaginal Arousal in NormalWomen

An important aspect of sexual motivation is physiological sexualresponding. Measured as an increase in vaginal vasocongestion elicitedby sexual stimuli, this responding is considered to be preparatory forcopulatory behavior¹⁰. In hypogonadotropic hypogonadal females we foundthat substitution with testosterone undecanoate 40 mg orally per dayduring an 8-week period enhanced vaginal responsiveness¹⁰. This effectwas not found in another group of hypogonadotropic hypogonadal patients(unpublished data). In both studies subjects received testosterone eachmorning, but patients in the first experiment were tested in theafternoon and patients in the second experiment in the morning. Thedifferent outcomes on physiological responding between these experimentsmay be caused by a time dependent effect of testosterone on vaginalarousal. In a third experiment, we examined whether administration of asingle dosage of testosterone sublingually, as compared with a placebo,increases vasocongestion during presentation of visual erotic stimuli¹¹.On treatment days we exposed eight sexually functional women withintervals of an hour and a half, to six erotic films depictingintercourse. The intake of testosterone caused a sharp increase inplasma levels of testosterone of short duration. About three to four anda half hours after this testosterone peak, we found a striking increasein vaginal responsiveness when the subjects were exposed to the visualsexual stimuli (see also FIG. 1). These findings demonstrate a time lagin the effect of sublingually administered testosterone on genitalarousal in sexually functional women.

The results of the above mentioned studies demonstrate that testosteroneis involved in female sexual motivation in a time dependent fashion. Theinfluence of sex steroids on sexual behavior might be explained by asteroid-responsive neural network, a highly interconnected group of sexhormone receptor-containing neurons in the brain¹². This network is nota closed circuit, but serves reproductive aims by functioning as anintegrating and activating center between external sensory cues,hormonal processes and reproductive behavior. This is partlyaccomplished by selective filtering of sensory input and amplificationof signals that may facilitate sexual behavior. We assume that anincrease in vaginal vasocongestion induced by sexual stimuli ispreparatory for copulatory behavior. Visual exposure to sexualintercourse between members of the species of the onlooker is a potentreleasing stimulus for such a preparatory motivational response. Bothmen and women have a marked capacity to respond to erotic films with agenital response¹³. The increased motivational sensitivity for sexualcues induced by testosterone is presumably the result from an alteredbrain state, in which dopaminergic, serotonergic and noradrenergicpathways are involved. This altered state might also be sensitive tointernal cues as evoked by sexual fantasy.

In another experiment we demonstrated that attention directed atalterations in genital arousal produced concordance betweenphysiological and subjective indices of sexual arousal¹⁴. There arereciprocal relationships between sexual desire, sexual arousal and theability and potency of orgasm. A reduced sexual desire will affectsexual arousal and visa versa; both indices of sexual function mightinfluence orgasm potency and visa versa.

Phosphodiesterase 5 Inhibitors and Sexual Arousal

In several studies it has been shown that selective type 5phosphodiesterase (PDE5) inhibitors improve erectile function in menwith erectile dysfunction, on average close to normal function¹⁵. In thepenis, nitric oxide (NO) released from nerves and endothelium, inducesproduction of cyclic guanosine monophosphate (CAMP). CAMP is a keymechanism in relaxing smooth muscle, necessary for the induction of anerection. This nucleotide is hydrolyzed by the phosphodiesterases, fromwhich the main activity in the corpora cavernosa is due to PDE5.Therefore, during sexual stimulation the action of NO/CAMP on erectilefunction will be enhanced by PDE5-inhibitors¹⁶. The genitalia of bothsexes have common embryological origins. Recently, it has been shownthat the clitoris consists of an erectile tissue complex, which embedsthe anterior vaginal wall. Clitoral erection and the anterior wall ofthe vagina are highly involved in female sexual arousal and response. Ithas recently been shown that sildenafil—a PDE5-inhibitor—improves sexualperformance in sexual functional women.¹⁷

Although in both men and women similar specialized vascular mechanismare involved in the genital response, an increase in Vaginal PulseAmplitude (VPA) cannot be considered to be the equivalent of anerection. A necessary but not sufficient condition for an erection isdilatation of arteries and resulting increased blood inflow. In thepenis there are corpora (corpus cavernosa (two) and corpus spongiosum(one)) containing small irregular compartments (vascular spaces). Thesmooth muscles in the cavernous sinusoidal walls are normally tonicallyconstricted under the control of an active sympathetic (adrenergic)tone. Relaxation of cavernous smooth muscle of the corpora results infilling and enlargements of the compartments with blood, which will beaccompanied by an erection. Although the precise mechanisms are unknown,sympathetic innervations and Nitric Oxide are believed to be bothprincipal mediators in relaxation of the corporeal smooth muscles. Inthe penis, sympathetic innervations of the blood vessels are sparse,while the smooth muscles are richly innervated by this system. Incontrast, the blood vessels of the penis are richly innervated by theparasympathetic system, while innervations of the smooth muscles by thissystem are sparse. Consequently, there are relatively independenteffects of these two parts of the peripheral nervous system on processesinvolved in the occurrence of an erection. Initiation of dilatation ofthe penile arteries and subsequent increase of blood flow to thecavernous tissue is regulated by the parasympathetic nervous system(initiation of an increase in this cholinergic activity depends onsignals by the brain). However, without relaxation of the smooth musclesthere will be no erection. Reduction of the sympathetic tone andconsequent relaxation of smooth muscles appears to be a relativeindependent prerequisite for the initiation of an erection. Thus, penileerection occurs in response to increased activity of the sacralparasympathetic innervations and a decreased activity of sympatheticpathways. In the penis, nitric oxide (NO) released from nerves andendothelium, induces production of cyclic guanosine monophosphate(CAMP). CAMP is a key mechanism in relaxing smooth muscle, necessary forthe induction of an erection. The production and release of NO might beinfluenced by a decrease in activity of the sympathetic branch.

Brain Activity Mediates the Influence of Psychosocial Circumstances onSexual Behaviour and Sexual Feelings via Inhibitory and ExcitatoryRegulating Mechanism

The prefrontal cortices of the human brain are crucial for cognitivefunctions involved in planning, execution and control of behaviour. Animportant aspect of these cognitive functions is inhibition of limbicsystem induced emotional responses, such as sexual behaviour.Psychological processes are also involved in three distinct(transitional and overlapping) phases of the human sexual response, aswell as disturbances in these phases leading to low sexual desire,sexual arousal problems and hampered orgasm. Thus, an increase inactivity of the prefrontal cortex associated with inhibition mightreduce sexual desire, sexual arousal and orgasm capacity. Activity inthe prefrontal cortices is also involved in the regulation of thesympathetic and parasympathetic branches of the peripheral nervoussystem. An increase in prefrontal activity is accompanied by a decreasein parasympathetic activity and an increase in sympathetic activity.Alterations in these branches occur asymmetrically. Moreover, thesympathetic branch of the peripheral nervous system seems to be moresensitive to psychogenic induced alterations. Thus psychologicalprocesses controlled by the prefrontal cortices appear to be directlyinvolved in the physiological mechanisms regulating induction of anerection. It might be assumed that the same physiological mechanisms areregulating the different components of the female sexual response (i.e.parasympathetic innervations are regulatory for the VPA, and sympatheticinnervations are, together with Nitric oxide, responsible for swellingand lubrication).

In a recently conducted experiment (not published) in healthy sexualfunctional women, we used a delayed measurement design in which subjectsingested one dosage of testosterone (0.5 mg sublingual) or placebo andafter 4 hours underwent fMRI while watching neutral and erotic videos.Hereafter subjects were conducted to a laboratory where their VPA inresponse to neutral and erotic videos was measured. As expected, in theplacebo condition we found activation of cortical and subcorticalstructures and deactivation of dorsal prefrontal areas comparable toother imaging studies on brain activation in response to eroticstimulation. Because of testosterone delayed effect on enhancing VPA, weexpected enhancement of these ‘erotic’ structures in this fMRI study.However, the opposite is true. Women on testosterone, show a decreasedactivation of all brain structures during erotic exposure implicated inthe normal sexual response. Furthermore, 2 structures showed a verysignificant increase: the septum, which functions as a restrainer ofemotional overshoot, and the left dorsolateral PFC, which functions asinhibitor of automated/reflexive responses. As stated, usuallydeactivation of dorsal prefrontal areas is observed.

Depending on circumstances and individual differences, testosterone canproduce effects that deviate from the expectations one would have giventhe functional role of testosterone in the regulation of sexualbehaviour. This inhibition of the centrally regulated autonomous sexualresponse was also apparent in the relative change in the VPA. Contraryto the expectations the VPA was smaller in the testosterone condition ascompared with placebo, and probably the result of a continuinginhibition mechanism induced during the fMRI procedure.

The Synergistic Effect of a Combination of Testosterone and PDE-5Inhibitor on Vaginal Arousal in Women Suffering from Female SexualDisorder

In a recently conducted experiment (see experimental part) in womensuffering from Female Sexual Dysfunction, we found at different timeintervals no effect from one dosage of testosterone, nor from aPDE5-inhibitor—as compared with a placebo—on vaginal arousability, noron sexual desire and genital sensations. In this experiment we found,however, that one dosage of testosterone (0.5 mg sublingual) combinedwith the administration of a PDE5-inhibitor (dosed in a way such that Tmax arises about 3.5-5.5 hours after the free testosterone peak) causedfour hours after the plasma testosterone peak a significant higherVaginal Pulse Amplitude during exposure to erotic stimuli. This effectwas less pronounced in a subgroup of women who were sexually abusedduring their childhood. We found no effect on sexual desire and onsubjective sexual arousal. Treatment with testosterone as well as thecombination of testosterone and a PDE 5 inhibitor caused an increase inattentional engagement (or withdrawal) for sexual cues, as compared withplacebo and/or a PDE5-inhibitor. Attentional engagement is an importantfunction for normal human sexuality. Testosterone combined with aPDE5-inhibitor produced a statistically significant increase in genitalarousal.

In one of its embodiments, the invention provides the use of acombination of a PDE5-inhibitor and testosterone or an analogue thereof,in the preparation of a medicament for the treatment of female sexualdysfunction.

Testosterone is also known under the chemical name17-β-hydroxyandrost-4-en-3-one which can be obtained in various ways: itmay be isolated and purified from nature or synthetically produced byany manner. The term “or an analogue thereof” includes any usefulmetabolite or precursor of testosterone, for example the metabolitedihydrotestosterone. It is clear to the skilled person that if ametabolite or precursor of testosterone is used, the time point foradministration of a PDE-5 inhibitor probably needs to be adapted. If,for example, dihydrotestosterone is used, the time of administration ofthe PDE5-inhibitor lies approximately half an hour earlier (as this isthe approximate time it takes for excess testosterone to be converted todihydrotestosterone). The amount of PDE5-inhibitors is still expandingand non-limiting examples are the following: GF-196960/IC351(tadalafil), Bay-38-9456 (vardenafil), UK-103320 (Sildenafil), E-4021,E-8010, E-4010, AWD-12-217 (zaprinast), AWD 12-210, UK-343,664,UK-369003, UK-357903, BMS-341400, BMS-223131, FR226807, FR-229934,EMR-6203, Sch-51866, IC485, TA-1790, DA-8159, NCX-911 or KS-505a. Otherexamples can be found in WO 96/26940.

Preferably, the PDE5-inhibitor is provided at least 3 hours after theadministration of the testosterone, even more preferably such thatC_(max) arises about 3.5 to 5.5 hours after the plasma free testosteronepeak. As already described above and depending on the formulation, thePDE5-inhibitor can also be given at the same time testosterone isadministrated.

Conditioning of Positive Associations Between Different Modalities ofthe Sexual Response

Treatments with a dosage of testosterone combined with a PDE5-inhibitorproduce alterations in brain and bodily functions which will makelearning of positive associations between sexual stimuli, genitalarousal and subjective experience possible. Moreover, the treatment ofFSD with a combination of testosterone and a PDE5-inhibitor ispreferably augmented by an “approach induction” treatment. To create amore permanent psychological change, the central en bodily processesactivated by testosterone and a PDE5-inhibitor under sexually relevantstimulation need to be perceived and need to become associated with apositive hedonic tone or with activation of the behavioral approachsystem. The perception of bodily reactions by focusing attention ongenital arousal is made possible by testosterone (whereby the genitalarousal is synergistically enhanced by the PDE5-inhibitor) and can beemphasized by verbal instructions. A positive hedonic tone cannot betaken for granted in the population of FSD patients. In order to achievea positive tone, patients can be exposed to positive stimuli during theeffective phase of the drugs (that is, at least 3 hours aftertestosterone intake). These positively motivated stimuli consist ofpictures of happy faces of persons of the patient's sexually preferredgender, possibly including the face of the partner. The pictures of thefaces are presented subliminally, so that in an unobtrusive way thebehavioral approach system becomes activated.

The treatment of FSD might consist of creating a situation in which thepatient learns to associate genital arousal with a positive hedonic toneor activation of the behavioral approach system. This requiresinducement of genital arousal (by sexual stimuli and a PDE5-inhibitor),sustained attention to sexual stimuli and to genital arousal (madepossible by testosterone) and activation of the behavioral approachsystem (by subliminal presentation of pictures of happy faces).

The invention further provides a method for treating a female sufferingfrom female sexual dysfunction by providing to said female a combinationof a PDE5-inhibitor and testosterone or an analogue thereof.

The invention will be explained in more detail in the following,non-limiting example.

EXAMPLES Participants

Fourteen women with a heterosexual orientation (Mean age: 40.6 years;sd: 10.4; premenopausal n=8, postmenopausal n=6) who have beenexperiencing FSD (i.e. low sexual desire, low sexual arousal ordecreased orgasm potency) for at least six months prior to study entryparticipated in this study. Subjects in fertile age used contraceptives(UID's, sterilization, oral contraceptives, except contraceptivescontaining anti-androgens). A pregnancy test was part of the procedure.Subjects were interviewed and examined by a gynecologist to excludepregnancy or breast feeding, vaginal infections, major operations to thevagina and/or vulva or unexplained gynecological complaints.Participants did not have a history of endocrinological, neurological ofpsychiatric treatment. Cardiovascular condition was tested and ECG waschecked for significant abnormalities. Standard blood chemistry andhematology tests were performed. Participants did not abuse drugs andwere required not to use alcohol or psychoactive drugs the eveningbefore and the day of experimentation. Subjects could not makeappointments during their period of menstruation.

Procedures

This study was approved by the Dutch medical-ethical committee(STEGMETC). The experimental trials were preceded by a screening visit.At this screening visit subjects were interviewed by apsychologist/gynecologist to diagnose for FSD and to determineeligibility for study participation. Weight, height, blood pressure(supine and standing), heart rate, respiration rate, and bodytemperature were measured. A supine, 12-lead ECG was recorded andexamined by a physician (and when necessary, a cardiologist). Agynecological examination and urine pregnancy test were performed.Cultures were taken to exclude Chlamydia or Gonococcus infections.

Eligible subjects underwent a familiarization trial following thescreening. A trained female experimenter familiarized the subject withstudy requirements and procedures. This included the use of the vaginalphotoplethysmograph (a tampon-shaped device). The subjects viewed during5 minutes a neutral film fragment followed by an erotic film fragmentsof 5 minutes. Hereafter they practiced on a shortened version of theemotional Stroop task.

For the experimental trials we used a double blind randomly assignedplacebo controlled cross-over design with four drug conditions:

1) Placebo

2) PDE5-inhibitor

3) Testosterone (0.5 mg sublingual)

4) testosterone+PDE5-inhibitor (the two compounds were given at the sametime, but the PDE5-inhibitor was dosed/formulated such that the effectof the testosterone and the effect of the PDE 5-inhibitor at leastpartly overlapped) Each subject underwent the four different drugtreatments (i.e. PDE5i, testosterone, PDE5i+testosterone and placebo) atfour separate experimental days. The four experimental days wereseparated by (at least) a three-day period.

During each day of drug manipulation subjects underwent the followingmeasurements:

−1:00 hrs: Emotional Stroop Task

−0:15 hrs: Trial 1

0:00 hrs: Intake drugs

0:05 hrs: Trial 2

2:45 hrs: Trial 3

4:15 hrs: Trial 4

Each day started with a physical examination (measurements of vitalsigns, blood pressure, heart rate, temperature and respiration rate).Blood samples (8 ml) were taken for hormone analyses. Subjects then wereseated in the sound attenuated, dimly lit experimental room. In order tomake this room less “sterile” geographic posters were hung on the wallsand an air perfumer was placed behind the subjects. The subjects thenexecuted the emotional Stroop task (15 minutes). The experimenterbrought the vaginal probe and the subject was left alone in the room toinsert the probe. The subject was instructed to sit as quietly aspossible while viewing the film fragments. A 10 minute neutral fragmentwas followed by a 5 minute erotic film fragment. After these baselinemeasurements, the subjects took medication: testosterone (0.5 mg; orplacebo) sublingually, with cyclodextrines as carrier and Vardenafil (10mg, or placebo) hidden in a capsule. The medication was followed byanother set of neutral (5 minutes) and erotic (5 minutes) filmfragments. The subjects removed the vaginal probe, and were taken to awaiting room for a pause of 2 hours. During this pause they couldconsume their lunch. Coffee and tea were unlimited available. This pausewas followed by a third set of neutral (5 minutes) and erotic (5minutes) film fragments. Again subjects had to wait for 2 hours, so thatthe last VPA measurement was taken 4 hours after medication. This lastfilm-trial (neutral, erotic film fragments) was followed by a secondpresentation of the emotional Stroop. The experimental day ended with ashort physical examination including drawing of a blood sample (8 ml forhormone analyses) and collection of AE and SAE.

FIG. 2 shows the effect of the pharmaceutical preparation according tothe invention (P<0.035) as compared with a placebo/PDE5-inhibitor on VPAin women suffering from FSD.

FIG. 3 shows the effect of the pharmaceutical preparation according tothe invention (P<0.04) as compared with a placebo, a PDE5-inhibitor andtestosterone in women suffering from FSD.

Emotional Stroop Task:

An unmasked and a masked version of the Emotional Stroop Task comparingcolour-naming latencies on neutral and erotic words were used. In boththe unmasked and the masked condition eight erotic and neutral words arepresented in different colours (i.e. red, green, blue and yellow). Anextra set of stimuli consisting of letter strings was used for practicetrials. Subjects were instructed to ignore the content of the words andto name the colour of the words as quickly as possible (in the maskedcondition, the colour of the mask has to be named). Each trial consistedof a fixation point which is shown for 750 ms, followed by the targetstimulus (the coloured neutral or erotic word). In the masked conditionthe word picture is presented for about 24 ms and then masked byrandomly cut, reassembled letters in the same colour. A microphoneconnected to a voice-level detector was placed in front of the subject.Initiation of vocal response was registered by the computer's clock andterminated the target presentation (with a no-response maximum of 3000ms). Thirty-two neutral words and thirty-two erotic words were presentedblocked. The same words were used for each test, however, the sequenceof words and colours differed all 8 times this task was used.

Results VPA Measurements

During the testosterone condition, one subject became nauseated watchingthe erotic film and decided not to participate further that day. For thepsychophysiological evaluation the VPA (Vaginal Pulse Amplitude) isused. The VPA reflects phasic changes in the blood volume correspondingwith each heartbeat; higher levels indicate higher levels of blood flow.The dependent variable used is the amplitude of the pulse wave. Beforethe mean VPA was calculated, the raw signal (sample rate was 20 Hz) wasdigitally bandpass filtered with a butterworth filter (−3 dB cutofffrequency range 0.7-1.5 Hz; 40 dB down/octave). Movement artefacts weredetected by visual inspection of the signal and removed manually.Hereafter the amplitude was measured as the distance between the top andbottom of a pulse wave. The mean VPA was calculated as the average ofthese amplitudes across 30 s periods.

The magnitude of the VPA response also depends on the exact placement ofthe probe. In order to compare conditions, it is not meaningful toexamine the absolute mean amplitude values in the neutral or eroticconditions. Instead, we use the mean change in amplitude from neutral toerotic film fragment divided by mean score in the neutral conditionduring each film trial as endpoint in the analyses.

The resulting difference scores were subjected to a 2 Steroid(testosterone yes/no)×2 PDE5i (vardenafil yes/no)×2 before-after(trial 1versus trial 4) repeated measures ANOVA. An interaction effect was foundfor the steroid condition over trials (F(1,12)=5.75; p<0.04) implicatingthat the increase from before to after measurement was significantlyhigher for drug conditions containing the steroid (testosterone+placeboand testosterone+Vardenafil) than for the drug conditions without thesteroid (placebo+placebo and placebo+Vardenafil). Further analyses ofthe before medication (trial 1) versus after medication (trial 4)responses showed that the increase in VPA between neutral and eroticfilms was not significant in the placebo condition nor in the Vardenfilor testosterone condition (see also FIG. 4). Only the combined treatmentcondition (Vardenfil+testosterone) lead to a statistically significantincrease in VPA before compared with after medication (F(1,12)=3.229;p=0.007).

We hypothesized the effects of sexual stimulation and medication ongenital responses to be associated with changes in central mechanisms.To test for associations with changes in attentional processes we usedthe StroopRT as measurement point. StroopRT is the difference in meanreaction times on colour naming of the neutral and the sexual words.

During visual inspection of the data, we found two subgroups within oursample: a group of women who were sexually abused during childhood and agroup of women who did not report such abuse. We decided to includesexual abuse during childhood as a between subjects variable in theanalyses.

In the unmasked condition no statistically significant results werefound. Analyses reported heretoforeward reflect results in the maskedcondition of the Stroop task. The MANOVA revealed a significantinteraction between Steroid, Trial and Sexual-abuse-in-childhood(F(2,10)=13.6; p=0.001). Within-subjects contrasts confirmed significanteffects for both VPA (F(1,11)=10.97; p=0.007) and StroopRT(F(1,11)=5.85; p=0.034) (FIG. 5).

Subjects who have been sexually abused during childhood show anattenuated VPA to erotic stimuli (before medication increase in VPAduring erotic film relative to neutral film is <60%, compared to >90%increase for not-abused subjects) and no increase over trials with orwithout testosterone. However, these subjects are not insensitive totestosterone. Under influence of testosterone they develop anattentional bias away from sexual stimuli (StroopRT increases).Not-abused subjects show an opposite pattern: their attention for sexualstimuli increases (StroopRT decreases) and parallel their VPA responseto the erotic film increases in the testosterone versus no-testosteroneconditions. (Univariate analyses: VPA: 2 Steroid (testosterone yes/no)×2Trial (before-after medication) ANOVA with VPA as within subjectsdependent measure and sexual abuse as between subjects factor:F(2,10)=5.9; p=<0.035; StroopRT: 2 Steroid (testosterone yes/no)×2 Trial(before-after) ANOVA with StroopRT as dependent measure and sexual abuseas between subjects factor: F(2,10)=4.2; p<0.07).

In this group of women suffering from FSD, Vardenafil did not lead to asignificant difference in VPA under condition of sexual stimulationcompared to placebo. Apparently, peripheral manipulations are notsufficient and central mechanisms need to be influenced. We show thattestosterone is such a centrally acting influence on sexual mechanisms.However, for our subjects, testosterone alone was not sufficient for asignificant increase in VPA compared to placebo. Only the combination oftestosterone and Vardenafil lead to a significant increase in VPAcompared with placebo.

We found that we could distinguish subgroups of subjects. For a subgroupof sexually abused women testosterone had an effect on attention forsexual stimuli. When these subjects had a greater attention for sexualstimuli their genital response did not increase. Both healthy women andwomen with FSD who are not abused respond with a genital reaction whentheir attention is directed to sexual stimuli. We showed in this studythat for patient without a history of abuse, the combination oftestosterone and a PDE5-inhibitor is beneficial in that it increasesboth resources allocated to the processing of sexual stimuli as well astheir genital response to these sexual stimuli.

The observed effect was less pronounced in a subgroup of women who weresexually abused during their childhood. For these women, thetestosterone or an analogue thereof and a PDE5-inhibitor is optionallycomplemented with psychotherapeutic intervention.

In a small study (N=4) we further investigated the efficacy ofsubliminal presentation of faces of happy men during erotic film clipsin 4 healthy women, following administration of the testosterone andPDE5-inhibitor combination. Women reported increased subjective sexualarousal in the subliminal happy male face presentation condition, incomparison to erotic film excerpts only condition.

FIGURES

FIG. 1

Graphs depicting a delay between testosterone administration and VPA inhealthy subjects

FIG. 2

Results of VPA measurement in women suffering from FSD receiving aplacebo combined with a PDE-5 inhibitor or the pharmaceuticalpreparation according to the present invention comprising testosteroneand a PDE-5 inhibitor (this last combination is also called “Lybrido”).

FIG. 3

Results of VPA measurement in women suffering from FSD receiving aplacebo, and PDE-5 inhibitor, testosterone or the pharmaceuticalpreparation according to the present invention.

FIG. 4

VPA measurements of different treatments

FIG. 5

Sexual abuse, attentional bias and VPA.

REFERENCES

-   -   1. Laumann, E. O., Paik. A. and Rosen, R. C.: Sexual dysfunction        in the United States: prevalence and predictors. JAMA 10: 281,        537, 1999.    -   2. Ikemoto, S. & Panksepp J. The role of nucleus accumbens        dopamine in motivated behavior: a unifying interpretation with        special reference to reward-seeking. Br Res Rev 31: 6-41, 1999.    -   3. Melis, M. R. & Argiolis, A. Dopamine and sexual behavior.        Neurosc. Biobehavioural Reviews 19: 19-38, 1995.    -   4. McCarthy M M, Albrecht E D. Steroid regulation of sexual        behavior. Trends Endocrinol Metab. 1996; 7:324-327.    -   5. Pfaff D W, Schwatz-Giblin S, McCarthy M M, Kowl L-M. Cellular        and molecular mechanisms of female reproductive behaviors. In:        Knobil E, Neill J D, eds. The Physiology of Reproduction, 2nd        ed, vol 2. New York, N.Y.: Raven Press; 1994: 107-220.    -   6. Freeman L M, Rissman E F. Neural aromatization and the        control of sexual behavior. Trends Endocrinol Metab. 1996;        7:334-337.    -   7. Waxenberg S E, Drellich M G, Sutherland A M. Changes in        female sexuality after adrenalectomy. J Clin End Metab. 1959;        19:193-202.    -   8. Drellich M G, Waxenberg S E. Erotic and affectional        components of female sexuality. In: Masserman J, ed. Science and        psycho-analysis. New York: Grune and Stratton, 1966.    -   9. Sherwin B B, Gelfland M M & Brender W: Androgens enhances        sexual motivation in females: a prospective, cross-over study of        sex steroid administration in the surgical menopause.        Psychosomatic Medicine, 49, 397, 1985.    -   10. Tuiten A, Laan E, Panhuysen G, Everaerd W, de Haan E,        Koppeshaar H & Vroon P: Discrepancies between genital responses        and subjective sexual function during testosteron substitution        in women with hypothalamic amenorrhea. Psychosomatic Medicine,        58, 234, 1996.    -   11. Tuiten A, van Honk J, Koppeschaar H, Bernaards C, Thijssen J        & Verbaten R: Time course of effects of testosterone        administration on sexual arousal in women. Archives of General        Psychiatry, 2000, 57, 149-154.    -   12. Cottingham S L, Pfaff D. Interconnectedness of steroid        hormone-binding neurons: existence and implications. Curr Topics        Neuroendocrinol 1986; 7:223-249.    -   13. Bancroft, J. Human Sexuality and its Problems (Churchill        Livingstone, Edinburg London Melbourne and New York, 1989).    -   14. Tuiten A, van Honk J, Verbaten R, Laan E, Everaerd W,        Stam H. Can Subliminal Testosterone increase subjective and        physiological measures of laboratory-induced sexual arousal?        Archives of General Psychiatry, 2002, 59, 465-466.    -   15. Potempa, A J., Bernard I., and Ulbrich E. Under Flexible        dosing, “Real world” condition PDE5 inhibitor improved erectile        function in a broad population of men. Europ Urol Suppl. 2:        96, 2003. Klotz T., Sashe R., Heidrich A., et al. PDE5 inhibitor        increases penile rigidity and tumescence in erectile dysfunction        patients: a Rigiscan and pharmacokinetic study. World J Urol 19:        32-39.

1. A kit of parts consisting essentially of a set of instructions fortreating a sexual disorder in a female, a first pharmaceuticalcomposition comprising testosterone or dihydrotestosterone (DHT),wherein said first pharmaceutical composition is provided in the form ofa sublingual formulation further comprising cyclodextrin and is designedto release all of the testosterone or DHT essentially immediately, and asecond pharmaceutical composition comprising a PDE5-inhibitor, whereinsaid second composition is designed for immediate release of saidPDE5-inhibitor, and wherein said instructions direct a subject toadminister the second composition 1.5-4.5 hours after administering thefirst composition.
 2. The kit of parts of claim 1, wherein the firstpharmaceutical composition comprises from 0.3 mg testosterone to 2.5 mgtestosterone.
 3. The kit of parts of claim 1, wherein the secondpharmaceutical composition comprises from 5 mg to 20 mg vardenafil. 4.The kit of parts of claim 1, wherein the second pharmaceuticalcomposition comprises from 25 mg to 100 mg sildenafil.
 5. The kit ofparts of claim 1, wherein the second pharmaceutical compositioncomprises from 5 mg to 20 mg tadalafil.
 6. The kit of parts of claim 1,further comprising a compound competing with testosterone for sexhormone binding globulin (SHBG) binding.
 7. The kit of parts of claim 1,wherein said testosterone or DHT provides a peak level in the patient'sblood circulation of free testosterone or DHT of at least 0.010 nmol/l.8. A method of treating a sexual disorder in a female, said methodcomprising: administering the kit of parts of claim 1 to a femalesuffering from said sexual disorder.
 9. A pharmaceutical formulationcomprising testosterone or DHT and a PDE5 inhibitor, wherein saidtestosterone provides a peak level in the patient's blood circulation offree testosterone or DHT of at least 0.010 nmol/l.